Role of Inositol Trisphosphate Receptors in Autophagy in DT40 Cells

Abstract

Previous studies have shown that small interfering RNA knockdown and pharmacological inhibition of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) stimulate autophagy. We have investigated autophagy in chicken DT40 cell lines containing targeted deletions of all three IP(3)R isoforms (triple knock-out (TKO) cells). Using gel shifts of microtubule-associated protein 1 light chain 3 as a marker of autophagy, we find that TKO cells have enhanced basal autophagic flux even under nutrient-replete conditions. Stable DT40 cell lines derived from TKO cells containing the functionally inactive D2550A IP(3)R mutant did not suppress autophagy in the same manner as wild-type receptors. This suggests that the channel function of the receptor is important in its regulatory role in autophagy. There were no marked differences in the phosphorylation state of AMP-activated protein kinase, Akt, or mammalian target of rapamycin between wild-type and TKO cells. The amount of immunoprecipitated complexes of Bcl-2-Beclin-1 and Beclin-1-Vps34 were also not different between the two cell lines. The major difference noted was a substantially decreased mTORC1 kinase activity in TKO cells based on decreased phosphorylation of S6 kinase and 4E-BP1. The discharge of intracellular stores with thapsigargin stimulated mTORC1 activity (measured as S6 kinase phosphorylation) to a greater extent in wild-type than in TKO cells. We suggest that basal autophagic flux may be negatively regulated by IP(3)R-dependent Ca(2+) signals acting to maintain an elevated mTORC1 activity in wild-type cells and that Ca(2+) regulation of this enzyme is defective in TKO cells. The protective effect of a higher autophagic flux in cells lacking IP(3)Rs may play a role in the delayed apoptotic response observed in these cells.